Probiotics, or beneficial bacteria, are promising agents for the prevention and treatment of pediatric inflammatory bowel disease (IBD). Several Lactobacillus species are considered to be probiotic organisms with attractive immunoregulatory features and are being consumed as nutritional supplements or alternative medicines. The proposed studies aim to comprehend basic mechanisms of immunoregulation by probiotics so that natural or engineered probiotics can be rationally devised for future medical therapies. The overall hypothesis is that probiotic Lactobacillus reuteri strains release oligosaccharides that suppress human tumor necrosis factor-alpha (TNF-a) by transcriptional regulation. Lactobacillus reuteri represents a model probiotic organism because this species has an impeccable safety record and has demonstrated beneficial effects in human individuals and mouse IBD models. L. reuteri is an established probiotic species that represents one of a select few indigenous Lactobacillus species of the human intestine. The specific aims of the proposal are as follows. 1) Investigate the relative abilities of Lactobacillus reuteri-derived oligosaccharides to inhibit human TNF-a by transcriptional regulation in primary human macrophages. Probiotic-derived oligosaccharide-enriched HPLC fractions from probiotic strains will be tested for TNF-inhibitory activity using primary cells derived from children with active Crohn's disease. 2) Explore suppression of AP-1-mediated transcriptional regulation of human TNF-a and modulation of MAP kinase signaling pathways in macrophages by probiotic oligosaccharides. Human macrophage models (two cell lines) that depend on either Toll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) signaling will be used to examine how probiotic oligosaccharides modulate MAP kinase signaling pathways. The identification of human signaling pathways regulated by probiotics will facilitate specification of targets for new anti-inflammatory compounds and may move probiotics from "alternative" to "mainstream" medicine. [unreadable] [unreadable] [unreadable]